Ultrasound-Assisted Synthesis, Antifungal Activity against Fusarium

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Ultrasound-Assisted Synthesis, Antifungal Activity against Fusarium oxysporum, and Three-Dimensional Quantitative Structure−Activity Relationship of N,S‑Dialkyl Dithiocarbamates Derived from 2‑Amino Acids Diego Quiroga,* Lili Dahiana Becerra, and Ericsson Coy-Barrera

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Bioorganic Chemistry Laboratory, Facultad de Ciencias Básicas y Aplicadas, Universidad Militar Nueva Granada, Campus Nueva Granada, 250247 Cajicá, Colombia ABSTRACT: A high-yielding, green, and fast synthesis of alkyl 2-substituted {[(alkylsulfanyl)carbonothioyl]amino}acetate-type compounds is described. The one-pot, threecomponent condensation of alkyl 2-aminoesters, carbon disulfide, and electron-deficient olefins was the key reaction to be developed. The products were obtained easily and efficiently, with good overall yields after two steps (79−91%), employing short reaction times, without the use of a catalyst, and ultrasonic irradiation in water. This procedure was exploited to produce antifungals against the phytopathogenic fungus Fusarium oxysporum. Some synthesized compounds exhibited good performance as mycelial growth inhibitors (IC50 < 80 μM). Structural and antifungal datasets were integrated to explore the comprehensive three-dimensional quantitative structure−activity relationship (3D-QSAR) using comparative molecular field analysis (CoMFA) and explain the observed activity. This integration resulted in an excellent CoMFA model (r2 = 0.812; q2 = 0.771) after substructure-based alignment. According to this model, synthesized compounds possessing steric bulky electronwithdrawing groups in the dithiocarbamate moiety can be considered as promising F. oxysporum inhibitors.

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INTRODUCTION Infectious episodes caused by fungal crop pests are an important, permanent threat to global food security.1,2 This threat is on the increase as a result of emerging problems associated with the increasing world population and global climate change, which encourage action to ensure that the right balance between high-volume food crops and excellent quality.3 Thus, the need to discover compounds with antifungal properties has increased intensely, but the employment and efficacy of such chemical agents are usually limited owing to toxicity, undesired side effects, application drawbacks in the field, pharmacokinetic problems, fungal resistance, and effectiveness on only one molecular target.4 Consequently, safer, efficient, novel, potent antifungals against phytopathogens with diverse physicochemical properties, low toxicity, and fewer side effects to the environment and human health are still required. The quantitative structure−activity relationship (QSAR) can exhibit significant performance in supporting the discovery and design of such chemical agents5 since it embodies the early steps toward a better interpretation of the structural requirements for bioactivity, even if the action mechanism can be unknown.6 Comparative molecular field analysis (CoMFA), the oldest genuine three-dimensional QSAR (3D-QSAR) approach, has been extensively applied to integrate molecular features (steric and electrostatic energies) and bioactivity datasets through statistical tools (e.g., partial least squares (PLS) regression).7 Thus, the main goals of such an integration are to assemble cross-validated models to © XXXX American Chemical Society

predict structure−activity observations and understand the essential molecular characteristics for promising antifungal activity.7 Several examples of CoMFA models for the discovery/design/development of antifungals against phytopathogens can be found in available publications with successful results comprising different scaffolds/moieties.5,6,8−10 N-Alkyl and N,N-dialkyl dithiocarbamates are part of an interesting group of sulfur compounds in chemistry. The importance of these compounds has increased recently due their potential applications related to materials/environmental science11−16 but also due to the broad band of biological activities of some metal-dithiocarbamate salts, such as antifungal, antitumoral, anticancer, and leishmanicidal activities,17−19 with the potential to be used in human health as well as crop protection. In fact, several examples of these dithiocarbamates (used as salts to ensure water solubility) have been marketed for a long time as fungicides, such as maneb, propineb, zineb, among others.20 The Fungicide Resistance Action Committee (FRAC) reports these kinds of complexes (FRAC code M03) with low risk of resistance since they have a multisite antifungal action.21 However, this action requires high concentrations of these active principles to be effective in crop protection, resulting in the main disadvantage Received: April 16, 2019 Accepted: July 25, 2019

A

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comprised the synthesis of the N,S-dialkyl dithiocarbamatecontaining compound series 6 (Figure 1) having varied substitutions to investigate their antifungal activity against F. oxysporum mycelial growth. Comprehensive 3D-QSAR using CoMFA was also accomplished to rationalize the structural requirements and further optimize these kinds of structures as antiphytopathogenic agents.

owing to the plausible ecotoxicological hazards related to metal accumulation in soils, food, and even water.22 Therefore, metal replacement in dithiocarbamate salts by an organic moiety to produce N,S-dialkyl dithiocarbamates has regularly been a matter of interest. In such a context, this metal substitution was already explored using natural polyols (e.g., glycerol), secondary amines, and carbon disulfide, affording moderate to good yields (55−93%),23 taking as base the previously reported one-pot, Michael-type addition of enones (or substitution of alkyl halides) to a dithiocarbamate moiety.24,25 Very recently, the design and antifungal action of a set of dithiocarbamic esters with an acrylate moiety were also reported, indicating that these compounds can be promising candidates for fungal control.26 From a green chemistry point of view, this approach has several economically and environmentally friendly factors to be highly attractive, such as using raw materials, being a metal-free process, and having 100% atom efficiency for sulfur and nitrogen.27 Using the above-mentioned approach, we have previously worked on dithiocarbamate functionalization as part of our research on organic synthesis toward novel antifungal agents against Fusarium oxysporum, a very problematic phytopathogen in several crops.28 Thus, a one-pot, efficient protocol for the synthesis of N,S-dialkyl dithiocarbamates 1 derived from Ltryptophan and electron-deficient olefins was previously reported under the search for indole-containing analogs as a strategy to obtain phytoalexin bioisosteres.29 Such a protocol involved an alkyl 2-aminoesters (from the respective 2-amino acid and aliphatic alcohols) instead of primary/secondary amines to add more elements to the green profile of this kind of N,S-dialkyl dithiocarbamate synthesis.27 In the present study, we expand our search for promising antifungal agents, performing the derivatization of the conventional 2-amino acids L-alanine 2, L-phenylalanine 3, and L-tyrosine 4 (Figure 1) using a similar strategy to widen the available chemical

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RESULTS AND DISCUSSION Synthesis. The synthetic route started with the conversion of 2−4 to the respective esters through a previously reported procedure.30 Thus, alkyl 2-aminoesters 5a−5l (Scheme 1)

Scheme 1. Synthesis of N,S-dialkyl Dithiocarbamates 6.1−6.36

were separately obtained in excellent yields (93−98%) from aliphatic alcohols (i.e., MeOH, EtOH, 2-PrOH, and n-BuOH). Thus, based on our background,29 reactions of 5a−5l with carbon disulfide and three Michael acceptors (i.e., acrylonitrile, methyl acrylate, and mesityl oxide) were carried out at room temperature, employing TEA as a base and THF as a solvent, during 24 h. Diluted hydrochloric acid was then added to this reaction mixture to remove TEA, and ethyl acetate was used for extracting the crude product. The main product was finally purified by means of conventional column chromatography (CC). 1H-NMR and ESI-MS showed the formation of 2substituted {[(alkylsulfanyl)carbonothioyl]amino}acetate-type compounds (6) in low yields (24−36%). An increase in the number of detectable products was observed when reactions were carried out at higher temperatures. These results suggested that these compounds could undergo side reactions under worse conditions such as reflux and strong acid or basic media. These reactions were explained, in turn, for Ltryptophan derivatives 1, which can undergo intramolecular cyclization, formation of N,N-dialkylthiourea as a side product, or C−S cleavage reactions.29 For an efficient methodology for the synthesis of compounds 6, sonochemical reaction principles were employed.31−34 So, all the reactions were carried out under ultrasound irradiation using TEA as a base and water as a solvent. After 1 h under irradiation at room temperature, compounds 6.1−6.36 were obtained in higher yields (87−92%) (Scheme 1), which were in accordance with previously reported results for other dithiocarbamate derivatives using primary and secondary amines (70−95%).24 However, upon performing reactions during longer times (>2 h) under ultrasonic irradiation, the

Figure 1. Chemical structures for compounds 1−6.

space to access optimized antifungal action. Additionally, as a contribution to the green synthesis of these N,S-dialkyl dithiocarbamates, the present protocol also includes a previously reported ultrasound-assisted approach for other dithiocarbamate derivatives,24 which importantly reduces the reaction time. Thus, this procedure can be considered attractive to synthesize these kinds of antifungal compounds since it is safer, nontoxic, cheaper, less time-consuming, and environment- and user-friendly. Thus, the aim of this work B

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Table 1. Antifungal Activity of Alkyl 2-Aminoesters (5a−5l) against F. oxysporum

IC50 values are mean ± standard deviation (SD) (n = 3). bpIC50(exp) = −log(IC50(exp) in M). cPredicted from the CoMFA model.

a

In vitro antifungal activity, directly tested on F. oxysporum mycelial growth sensitivity, was assessed through a 12-well plate amended-medium method39 using a F. oxysporum isolate from Cape gooseberry. Five concentrations (between 10 and 300 μg/mL) were separately used to evaluate the antifungal activity of all synthesized 5 and 6 series compounds. Thus, the half-maximal inhibitory concentration (IC50) values were determined, which are listed in Tables 1 and 2. IC50 data for all test compounds were found to be within the 0.056−6.0 mM range. Some commercially available fungicides exhibit activity on mycelial growth inhibition, with IC50 values of ca. 100 μg/ mL.40,41 The present study included two commercial antifungals, that is, mancozeb (a Mn/Zn dithiocarbamate salt) and iprodione (an N-substituted hydantoin carboxamide). The IC50 values of these antifungals were 0.031 and 0.043 mM, respectively. These facts indicated that the activity exhibited by some synthetic N,S-dialkyl dithiocarbamate derivatives can therefore be considered promising (Table 2). In this sense, the results also suggested a trend to enhance the antifungal activity against F. oxysporum upon performing structural modifications in the 2-amino acid skeleton. As expected, alkyl 2-aminoesters 5a−5l have low activity (IC50 = 2.1−6.0 mM range, Table 1), but their functionalization toward N,S-dialkyl dithiocarbamate-

number of detectable compounds increased considerably. This behavior can be understood considering the collateral effects of ultrasound on organic reactions. Cavitation phenomena can create and enlarge gaseous and vaporous cavities within the irradiated liquid medium. These cavities implode, inducing higher local temperatures and pressures inside the bubbles. This physical process enhances the molecular diffusion through the creation of a turbulent flow in the liquid medium.35 Although some reactions can be accelerated by ultrasonic irradiation, thermal unstable compounds, such as alkyl dithiocarbamates, can be decomposed under longer reaction times.36 Antifungal Activity of Synthetic Compounds. It is known that the dithiocarbamate functional group, in both linear and cyclic compounds, has important influence on the bioactivity of several agents.37 Specifically, antifungal activity results have demonstrated a good correlation between the variations on percent inhibition and the steric−electrostatic properties.26,38 To understand the influence of structural modifications in the dithiocarbamate skeleton with respect to the antifungal activity of the synthesized brassinin analog results, it is important to perform successful new bioactive agent designs. C

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Table 2. Antifungal Activity of Test N,S-Dialkyl Dithiocarbamates (6.1−6.36) against F. oxysporum

IC50 values are mean ± standard deviation (SD) (n = 3). bpIC50(exp) = −log(IC50(exp) in M). cPredicted from the CoMFA model.

a

D

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were then calculated using probes (steric and electrostatic ones), and after several steps of data pretreatment, partial least squares (PLS) regression was carried out (using five PLS components) to build linear relationships among variations in the MIF values as a function of changes in the experimental pIC50. After that, the best model required three PLS components, suggesting good correlation between MIF values and the experimental pIC50 of test compounds. A statistically reliable, predictive CoMFA model was then achieved from substructure-based alignment. Thus, the model resulted in a conventional correlation coefficient r2 = 0.812, the crossvalidated LOO coefficient q2 = 0.771, the cross-validated LMO coefficient q2 = 0.768, and the F-test value of 122.876, demonstrating excellent parameters for a robust model.48 After the Y-scrambling procedure (enlisting the observations randomly using 20 scramblings and 10 runs), there was no correlation between MIF and activity datasets; hence, the model decayed drastically. The mean Rscr2 and Qscr2 of the first 50 iterations were lower than those of the cross-validated model (Figure 2b), indicating that the model is good and it was not achieved as a result of a chance correlation.

type compounds (6.1−6.36) led to better IC50 values (Table 2). These results demonstrated the importance of the sulfurcontaining functional groups, that is, dithiocarbamates, as an important moiety/fragment in antifungal agents to reach plausible biological interactions and improve the fungal growth-related inhibition of test compounds.42 Several reports in the literature43,44 have proposed that this inhibition is due to the ability of the dithiocarbamate residues to be rapidly absorbed by the mycelium of the fungus. Then, isocyanates are generated, which react mainly with the thiol groups of enzymes and metabolites of the cells, causing alterations in the hyphae and interruptions in the supply of energy to the fungal cells. A nonimportant bulky influence on antifungal activity of substituent R2 in series 6 was also suggested from results. However, a bulky group (R1) at C2 in 2-amino acid moiety presumably reduces antifungal activity, which can vary slightly according to the 2-amino acid precursor. Compounds derived from L-alanine, 6.1−6.12, showed higher antifungal activity compared to compounds 6.13−6.36 derived from L-phenylalanine and L-tyrosine. The incidence of (CX)−R moiety (X = O, N; R = Me, OMe) in the R−S(CS) alkyl fragment can be discussed according to the obtained results. Within the compounds derived from L-alanine, compounds 6.1, 6.4, 6.8, and 6.11 showed the lowest IC50 values. These compounds are smaller and of lower molecular weight due to the size of the substituent R1, which probably makes these types of molecules more easily absorbed by the fungal cells, thus favoring their efficacy. The absorption of larger dithiocarbamates may be diminished due to an impediment to passing through the fungal wall and the plasma membrane. Additionally, complementary metabolic steps are needed to hydrolyze larger substituents. These compounds are differentiated by a nitrile group. The influence of the CN group as a hydrogen bond acceptor and its strong dipole (which enhances polar interactions) could explain our results upon considering the intermolecular binding interactions with key residues of the plausible active site in different functional target enzymes of phytopathogens.45 Within the compounds derived from L-phenylalanine and Ltyrosine, 6.13−6.36, the results showed a similar tendency for L-alanine derivatives. However, compounds 6.17 and 6.20 (obtained upon using methyl acrylate) also showed promising antifungal activity. The influence of the methoxycarbonyl fragment could improve the interaction within the active site of functional enzymes in the fungus metabolism due its electronic and structural characteristics.46 Comparative Molecular Field Analysis (CoMFA). The structure−antifungal activity relationship of synthetic compounds was reasonably explained by a three-dimensional quantitative structure−activity relationship (3D-QSAR) study through a comparative molecular field analysis (CoMFA), which was also performed. The dataset was expanded involving the close-related, previously synthesized N,S-dithiocarbamates from L-tryptophan29 in order to include a wider range of antifungal activity values of the training set compounds. A basic assumption in CoMFA models is that an appropriate building of the steric and electrostatic fields around a set of aligned molecules might provide all the information necessary for understanding their biological activities.47 Therefore, experimental antifungal data (as pIC50 values) and aligned structures were subdivided into a test set (30%) and a training set (70%) for external validation and generating the CoMFA model, respectively. The molecular interaction fields (MIFs)

Figure 2. (a) CoMFA predicted as experimental pIC50 values. (b) Plot of Y-scrambled Rscr2 and Qscr2 of each iteration (n = 50) compared with the cross-validated model (R2 and Q2).

According to this analysis, experimental and predicted activity data for the whole compound set (Tables 1 and 2), expressed as pIC50, and the corresponding plot (predicted versus the experimental pIC50 values) for the entire set of test compounds indicated good correlation for both training and test datasets (experimental) and the predicted activity values (theoretical) (Figure 2a). There was no evidence of outliers within the standard deviation (i.e., 0.203), having normal residual values. Thus, the predictability regarding the MIF values of promising antifungals can be considered adequate because it exhibited an appropriate fit for modeling the F. oxysporum mycelial growth inhibition of these kinds of compounds. Blue dots represent predictions for the training set; red dots represent predictions for the test set. The CoMFA model also involved contributions of steric and electrostatic E

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fields of 61 and 33%, respectively. Thus, particular contour surfaces of these field contributions (model stdev*coeff), including test compounds from the CoMFA analysis, are shown in Figure 3.

might also be favorable to increase the activity. From all of these facts, these structural features improve antifungal activity and should be considered in further structural optimization studies to develop more potent antifungals.

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CONCLUSIONS

In summary, we reported a high-yielding, green, and fast ultrasound-assisted synthesis of N,S-dialkyl dithiocarbamates derived from common 2-amino acids and electron-deficient olefins (Michael acceptors) under mild conditions to produce antifungal compounds against a problematic phytopathogen. Hence, the results for the in vitro antifungal activity against F. oxysporum and the respective 3D-QSAR analysis demonstrated the promising performance (IC50 < 80 μM) of the synthesized compounds as leads for the development of a novel class of F. oxysporum mycelial growth inhibitors based on this type of N,S-dialkyl dithiocarbamates. A relationship between the structure and antifungal activity was demonstrated, involving smaller side chains in alkyl 2-aminoester moieties (i.e., alanine) and a cyanoethyl substitution as an EWG, which can be rationalized by the efficient absorption ability through the wall of fungal hyphae, enhancing/favoring antifungal activity. From these facts, further studies are then required to discern the action mechanism of these antifungal compounds. The most active compound was found to be (S)-ethyl 2-((2cyanoethylthio)carbonothioylamino)propanoate (6.4) (IC50 = 56 μM), which can be considered as a promising antifungal agent to support the control of F. oxysporum as a crop pest.

Figure 3. (a) Steric and (b) electrostatic maps from the CoMFA model including test compounds. Green contours (32% contribution) show positions where a bulky group would be favorable for higher antifungal activity. Yellow contours (29% contribution) indicate positions where a decrease in the bulk of the target molecules is favored. Blue contours (18% contribution) encompass regions where an increase of positive charge will enhance the activity. Red contoured areas (15% contribution) indicate that more negative charges are favorable for the activity. (c) Scheme for the steric and electrostatic contributions according to the CoMFA model.

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MATERIALS AND METHODS General Information. Entire set of reagents and chemicals was commercially acquired (Merck KGaA and/or SigmaAldrich). They were employed without additional refinement. The purity of dry solvents was sufficiently defined during purchasing. Progression of reactions and purifications of products were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 plates (Merck KGaA) under detection at 254 nm. Ultrasound-assisted reactions were performed in an Elmasonic S30H ultrasonic bath with 37 Hz of frequency. Silica gel 60 (0.040−0.063 mm mesh) (Merck KGaA) was used for CC. A Bruker Avance AV-400 MHz spectrometer was employed for nuclear magnetic resonance (NMR) experiments. TMS was utilized as a reference to give chemical shifts in δ (ppm). Typical splitting patterns were implemented to define the signal multiplicity (i.e., s, singlet; d, doublet; t, triplet; m, multiplet). A Shimadzu Prominence HPLC module system was operated for HPLC runs, employing a L2 column (Synergi HydroRP-C18, Phenomenex, 150 mm × 4.6 mm × 4 μm) and a mobile phase containing a mixture of 0.1% formic acid in water (B) and 1% formic acid in acetonitrile (A), in gradient mode, at 1.5 mL/min (flow rate). A multiwavelength diode array detector was used for UV detection (270 nm). A LC−MS 2020 spectrometer (Shimadzu, Japan) was utilized for recording mass spectra in positive ion mode using electrospray ionization. A JASCO P-2000 polarimeter (JASCO Co., Ltd., Japan) in a quartz cell (1.0 cm) was used for determining optical specific rotations of synthetic compounds, expressed as mean values after 10 independent determinations. General Procedure for the Synthesis of Alkyl Esters 5a−5l. Compounds 5a−5l were prepared as previously illustrated30 after some modifications. To a solution of the

In the steric field contour map, sterically favorable and unfavorable regions are represented as green and yellow contours, respectively. The green contours show positions where a bulky group might favor higher antifungal activities, whereas the yellow contours indicate positions where a decrease in the bulk of the target molecules is favored. In this context, these contours let us determine that electronwithdrawing groups attached to sulfur on the dithiocarbamate moiety could be replaced by bulky groups to increase antifungal activity, as mentioned before regarding CN and COOMe substituents. The bulky substitution at R2 (esterforming alkyl group) did not favor antifungal activity, indicating a preference for nonbulky 2-aminoester precursors. In contrast, the activity was evidently favored when the 2amino acid-derived moiety had less bulky groups at R1 (amino acid side chain). Therefore, compounds 6.1, 6.4, 6.7, 6.8, and 6.11 fit very well for these structural requirements to be highly active as F. oxysporum mycelium growth inhibitors (IC50 < 80 μM). Furthermore, potential electrostatic interactions also correlate positively to antifungal activity involving desirable positive and negative partial charges (blue and red contours, respectively) in molecules to enhance the antifungal activity. These contours were found to be distributed into two faces of the aligned molecules. This can be rationalized suitably because electron-withdrawing groups (EWGs) (directly bonded to the dithiocarbamate moiety) evidently cause an electron-deficient zone in this molecule region. Hence, the presence of these negatively charged EWGs favored antifungal activity. Other blue contours over the nitrogen and aromatic side chain (R1) suggest that electron-deficient substituents F

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Methyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-L-alaninate (6.3). Yellow resin; yield, 81%; 1 H-NMR (400 MHz, CDCl3): δH 7.67 (s, 1H), 5.08 (q, 3J = 7.0 Hz, 1H), 3.76 (s, 3H), 2.25 (s, 2H), 1.69 (s, 3H), 1.53 (d, 3 J = 7.0 Hz, 3H), 1.41 (s, 3H), 1.34 (s, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.5, 22.5, 25.1, 30.32, 52.4, 52.8, 53.6, 169.5, 195.7, 208.6 ppm. ESI-MS m/z [M + H]+, 278.09. [α]25 D = +4.60 ± 0.346 (c 0.1, MeOH). Ethyl (((2-Cyanoethyl)thio)carbonothioyl)-L-alaninate (6.4). Yellow resin; yield, 89%; 1H-NMR (400 MHz, CDCl3): δH 7.67 (s, 1H), 5.08 (q, 3J = 7.1 Hz, 1H), 4.21 (q, 3 J = 7.0 Hz, 2H), 3.49 (t, 3J = 6.9 Hz, 2H), 2.92 (t, 3J = 6.9 Hz, 2H), 1.53 (d, 3J = 7.1 Hz, 3H), 1.29 (t, 3J = 7.0 Hz, 3H) ppm. 13 C-NMR (100 MHz, CDCl3): δC 14.3, 19.8, 25.3, 38.1, 54.4, 60.4, 118.9, 170.4, 194.8 ppm. ESI-MS m/z [M + H]+, 246.06. [α]25 D = −7.40 ± 0.800 (c 0.1, MeOH). Ethyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-Lalaninate (6.5). Yellow resin; yield, 82%; 1H-NMR (400 MHz, CDCl3): δH 7.59 (bs, 1H), 5.11 (q, 3J = 6.9 Hz, 1H), 4.24 (q, 3J = 7.1 Hz, 2H), 3.70 (s, 3H), 3.50 (t, 3J = 6.8 Hz, 2H), 2.76 (t, 3J = 6.8 Hz, 2H), 1.52 (d, 3J = 6.9 Hz, 3H), 1.29 (t, 3J = 7.1 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 14.1, 19.7, 25.2, 38.4, 51.5, 53.8, 59.7, 169.9, 171.7, 194.5 ppm. ESI-MS m/z [M + H]+, 280.07. [α]25 D = −31.6 ± 1.02 (c 0.1, MeOH). Ethyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-Lalaninate (6.6). Yellow resin; yield, 80%; 1H-NMR (400 MHz, CDCl3): δH 7.66 (s, 1H), 5.09 (q, 3J = 7.0 Hz, 1H), 4.24 (q, 3J = 6.9 Hz, 2H), 2.25 (s, 2H), 1.68 (s, 3H), 1.52 (d, 3J = 7.0 Hz, 3H), 1.41 (s, 3H), 1.36 (s, 3H), 1.23 (t, 3J = 6.9 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 14.2, 20.1, 24.9, 38.4, 50.8, 53.6, 60.9, 170.4, 172.3, 195.0 ppm. ESI-MS m/z [M + H]+, 292.10. [α]25 D = −1.43 ± 0.153 (c 0.1, MeOH). Isopropyl (((2-Cyanoethyl)thio)carbonothioyl)-L-alaninate (6.7). Yellow resin; yield, 90%; 1H-NMR (400 MHz, CDCl3): δH 7.67 (s, 1H), 5.07 (m, 1H), 4.82 (m, 1H), 3.49 (t, 3J = 6.9 Hz, 2H), 2.93 (t, 3J = 6.9 Hz, 2H), 1.53 (d, 3J = 7.1 Hz, 3H), 1.35 (d, 3J = 5.8 Hz, 6H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.5, 21.7, 25.0, 38.4, 54.5, 67.3, 119.2, 170.3, 194.9 ppm. ESI-MS m/z [M + H]+, 260.08. [α]25 D = +3.33 ± 0.231 (c 0.1, MeOH). Isopropyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)1 L-alaninate (6.8). Yellow resin; yield, 88%; H-NMR (400 MHz, CDCl3): δH 7.54 (bs, 1H), 5.11 (m, 1H), 4.82 (m, 1H), 3.70 (s, 3H), 3.50 (t, 3J = 6.9 Hz, 2H), 2.76 (t, 3J = 6.9 Hz, 2H), 1.52 (d, 3J = 7.0 Hz, 3H), 1.35 (d, 3J = 6.0 Hz, 6H) ppm. 13 C-NMR (100 MHz, CDCl3): δC 19.8, 22.0, 25.2, 38.9, 51.5, 53.7, 70.1, 169.9, 172.2, 195.1 ppm. ESI-MS m/z [M + H]+, 294.07. [α]25 D = +2.13 ± 0.416 (c 0.1, MeOH). Isopropyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-L-alaninate (6.9). Yellow resin; yield, 79%; 1 H-NMR (400 MHz, CDCl3): δH 7.67 (s, 1H), 5.08 (m, 1H), 4.82 (m, 1H), 2.26 (s, 2H), 1.68 (s, 3H), 1.53 (s, 3H), 1.41 (s, 3H), 1.34 (s, 3H), 1.25 (d, 3J = 5.9 Hz, 6H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.4, 21.9, 22.6, 25.3, 30.2, 52.9, 53.7, 68.2, 170.2, 196.1, 209.1 ppm. ESI-MS m/z [M + H]+, 306.11. [α]25 D = 2.54 ± 0.109 (c 0.1, MeOH). Butyl (((2-Cyanoethyl)thio)carbonothioyl)-L-alaninate (6.10). Yellow resin; yield, 89%; 1H-NMR (400 MHz, CDCl3): δH 7.67 (s, 1H), 5.08 (m, 1H), 4.18 (t, 3J = 6.6 Hz, 2H), 3.49 (t, 3J = 6.8 Hz, 2H), 2.92 (t, 3J = 6.8 Hz, 2H), 1.62 (m, 2H), 1.53 (d, 3J = 5.8 Hz, 3H), 1.36 (m, 2H), 0.94 (t,

respective amino acid (1 mmol) previously dissolved in the corresponding aliphatic alcohol (5.0 mL) was added dropwise trimethylsilane chloride (4 mmol). The resulting mixture was heated under reflux and stirred for 4 h. Subsequently, the heating was stopped, and this mixture was left until room temperature. Solvent was then removed by vacuum distillation. The crude reaction product was recrystallized from ethanol to obtain the respective hydrochloride. Compounds 5a−5l were obtained as their amine form by treatment with a 10% NaHCO3 and discontinuous liquid−liquid extraction with ethyl acetate (5 × 10 mL). The synthetic compounds were successfully characterized by electrospray-mass spectrometry (ESI-MS) in positive ion mode: methyl (2S)-2-aminopropanoate (5a), yield, 97%; m/z [M + H]+, 104.07; ethyl (2S)-2-aminopropanoate (5b), yield, 94%; m/z [M + H]+, 118.09; 2-propyl (2S)-2-aminopropanoate (5c), yield, 91%; m/z [M + H]+, 132.10; n-butyl (2S)-2-aminopropanoate (5d), yield, 89%; m/z [M + H]+, 146.12; methyl (2S)-2-amino-3phenylpropanoate (5e), yield, 93%; m/z [M + H]+, 180.1; ethyl (2S)-2-amino-3-phenylpropanoate (5f), yield, 92%; m/z [M + H]+, 194.12; 2-propyl (2S)-2-amino-3-phenylpropanoate (5g), yield, 87%; m/z [M + H]+, 208.13; n-butyl (2S)-2amino-3-phenylpropanoate (5h), yield, 89%; m/z [M + H]+, 222.15; methyl (2S)-2-amino-3-(4-hydroxyphenyl)propanoate (5i), yield, 97%; m/z [M + H]+, 196.10; ethyl (2S)-2-amino-3(4-hydroxyphenyl)propanoate (5j), yield, 94%; m/z [M + H]+, 210.11; 2-propyl (2S)-2-amino-3-(4-hydroxyphenyl)propanoate (5k), yield, 86%; m/z [M + H]+, 224.13; n-butyl (2S)-2-amino-3-(4-hydroxyphenyl)propanoate (5l), yield, 91%; m/z [M + H]+, 238.14. General Procedure for the Synthesis of N,S-dialkyl Dithiocarbamates 6.1−6.36. For method A, compounds 6.1−6.36 were synthesized using the described procedure in the literature.29 Conventional CC was employed for purifying the products. For method B via ultrasonic irradiation, a mixture of the corresponding alkyl esters 5a−5l (1 mmol), carbon disulfide (1 mmol), the corresponding Michael acceptor (1 mmol) (i.e., acrylonitrile, methyl acrylate, or mesityl oxide), and triethylamine (TEA, 1 mmol) in water (10 mL) was placed in a Pyrex round-bottom flask coupled to reflux. The reaction mixture was irradiated by ultrasound (37 kHz) using an Elmasonic E30H sonicator (Elma Schmidbauer GmbH, Germany) with an HF outpower of 240 W at room temperature during 1 h. TLC monitoring was implemented using ethyl acetate/petroleum ether mixtures as mobile phases. Reduced pressure was used for concentrating the reaction mixtures. Conventional CC was employed for purifying the afforded products. Methyl (((2-Cyanoethyl)thio)carbonothioyl)-L-alaninate (6.1). Yellow resin; yield, 91%; 1H-NMR (400 MHz, CDCl3): δH 7.68 (s, 1H), 5.08 (q, 3J = 7.1 Hz, 1H), 3.76 (s, 3H), 3.48 (t, 3J = 7.0 Hz, 2H), 2.91 (t, 3J = 7.0 Hz, 2H), 1.53 (d, 3J = 7.1 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.5, 25.1, 38.2, 53.1, 54.3, 119.1, 170.2, 195.1 ppm. ESI-MS m/z [M + H]+, 232.05. [α]25 D = −2.00 ± 0.200 (c 0.1, MeOH). Methyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-Lalaninate (6.2). Yellow resin; yield, 85%; 1H-NMR (400 MHz, CDCl3): δH 7.65 (s, 1H), 5.11 (q, 3J = 7.1 Hz, 1H), 3.76 (s, 3H), 3.70 (s, 3H), 3.50 (t, 3J = 6.9 Hz, 2H), 2.76 (t, 3J = 6.9 Hz, 2H), 1.52 (d, 3J = 7.1 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.8, 25.1, 38.6, 51.4, 52.9, 53.8, 170.1, 172.0, 194.7 ppm. ESI-MS m/z [M + H]+, 266.05. [α]25 D = −2.67 ± 0.416 (c 0.1, MeOH). G

DOI: 10.1021/acsomega.9b01098 ACS Omega XXXX, XXX, XXX−XXX

ACS Omega

Article

J = 7.4 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 13.8, 19.3, 19.5, 25.1, 30.9, 38.2, 54.3, 64.4, 119.1, 170.2, 195.1. ESIMS m/z [M + H]+, 274.09. [α]25 D = +1.27 ± 0.115 (c 0.1, MeOH). Butyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-Lalaninate (6.11). Yellow resin; yield, 86%; 1H-NMR (400 MHz, CDCl3): δH 7.63 (s, 1H), 5.12 (m, 1H), 4.17 (t, 3J = 6.4 Hz, 2H), 3.69 (s, 3H), 3.49 (t, 3J = 7.1 Hz, 2H), 2.91 (t, 3J = 7.1 Hz, 2H), 1.69−1.58 (m, 2H), 1.52 (d, 3J = 6.0 Hz, 3H), 1.43−1.29 (m, 2H), 0.92 (t, 3J = 7.4 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 13.7, 19.4, 20.1, 25.2, 31.1, 38.6, 51.2, 53.7, 64.5, 170.1, 171.8, 195.1 ppm. ESI-MS m/z [M + H]+, 308.10. [α]25 D = −18.8 ± 1.11 (c 0.1, MeOH). Butyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-Lalaninate (6.12). Yellow resin; yield, 79%; 1H-NMR (400 MHz, CDCl3): δH 7.67 (s, 1H), 5.13−5.03 (m, 1H), 4.18 (t, 3J = 6.6 Hz, 2H), 2.28 (s, 2H), 1.69 (s, 3H), 1.65−1.60 (m, 2H), 1.53 (s, 3H), 1.42 (s, 3H), 1.36 (d, 3J = 5.8 Hz, 3H), 1.19 (m. 2H), 0.94 (t, 3J = 7.3 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 13.8, 19.3, 19.7, 22.7, 25.1, 30.2, 31.1, 52.5, 52.9, 53.5, 65.0, 170.1, 196.0, 207.6 ppm. ESI-MS m/z [M + H]+, 320.14. [α]25 D = +6.00 ± 0.693 (c 0.1, MeOH). Methyl (((2-Cyanoethyl)thio)carbonothioyl)-L-phenylalaninate (6.13). Yellow resin; yield, 91%; 1H-NMR (400 MHz, CDCl3): δH 7.54−7.04 (m, 5H), 5.44−5.37 (m, 1H), 3.74 (s, 3H), 3.46−3.35 (dd, 2J = 14.0 Hz, 3J = 6.2 Hz, 1H), 3.19−3.12 (dd, 2J = 14.0 Hz, 3J = 6.2 Hz, 1H), 2.93 (t, 3J = 6.8 Hz, 2H), 2.84 (t, 3J = 6.8 Hz, 2H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.5, 37.2, 38.2, 53.1, 54.3, 119.1, 129.9, 130.2, 136.3, 170.2, 195.1 ppm. ESI-MS m/z [M + H]+, 308.08. [α]25 D = +5.53 ± 1.21 (c 0.1, MeOH). Methyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-Lphenylalaninate (6.14). Yellow resin; yield, 91%; 1H-NMR (400 MHz, CDCl3): δH 7.50−7.08 (m, 5H), 5.47−5.40 (m, 1H), 3.75 (s, 3H), 3.71 (s, 3H), 3.50 (t, 3J = 7.2 Hz, 2H), 3.40 (dd, 2J = 13.9 Hz, 3J = 6.2 Hz, 1H), 3.21 (dd, 2J = 13.9 Hz, 3J = 6.2 Hz, 1H), 2.76 (t, 3J = 7.2 Hz, 2H) ppm. 13C-NMR (100 MHz, CDCl3): δC 20.1, 36.9, 38.4, 51.1, 52.9, 53.8, 130.2, 131.5, 136.8, 170.6, 172.5, 194.4 ppm. ESI-MS m/z [M + H]+, 342.08. [α]25 D = +6.13 ± 0.643 (c 0.1, MeOH). Methyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-L-phenylalaninate (6.15). Yellow resin; yield, 81%; 1H-NMR (400 MHz, CDCl3): δH 7.51−7.07 (m, 5H), 5.46−5.41 (m, 1H), 3.75 (s, 3H), 3.32 (dd, 2J = 13.9 Hz, 3 J = 6.2 Hz, 1H), 3.10 (dd, 2J = 13.9 Hz, 3J = 6.2 Hz, 1H), 2.17 (s, 2H), 1.63 (s, 3H), 1.25 (s, 3H), 1.11 (s, 3H) ppm. 13CNMR (100 MHz, CDCl3): δC 19.7, 22.9, 30.2, 37.4, 52.6, 52.9, 53.4, 129.7, 130.1, 136.4, 170.2, 196.1, 207.9 ppm. ESI-MS m/ z [M + H]+, 354.12. [α]25 D = +8.33 ± 1.10 (c 0.1, MeOH). Ethyl (((2-Cyanoethyl)thio)carbonothioyl)-L-phenylalaninate (6.16). Yellow resin; yield, 89%; 1H-NMR (400 MHz, CDCl3): δH 7.54−7.09 (m, 5H), 5.44−5.37 (m, 1H), 4.20 (q, 3 J = 7.1 Hz, 2H), 3.40 (dd, 2J = 14.0 Hz, 3J = 6.2 Hz, 1H), 3.16 (dd, 2J = 14.0 Hz, 3J = 6.2 Hz, 1H), 2.94 (t, 3J = 6.8 Hz, 2H), 2.84 (t, 3J = 6.8 Hz, 2H), 1.26 (t, 3J = 7.1 Hz, 3H) ppm. 13CNMR (100 MHz, CDCl3): δC 14.3, 19.8, 37.2, 38.1, 54.4, 60.4, 118.9, 129.9, 130.2, 136.3, 170.4, 194.8 ppm. ESI-MS m/z [M + H]+, 322.09. [α]25 D = +15.2 ± 1.06 (c 0.1, MeOH). Ethyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-Lphenylalaninate (6.17). Yellow resin; yield, 91%; 1H-NMR (400 MHz, CDCl3): δH 7.52−7.09 (m, 5H), 5.45−5.40 (m, 1H), 4.20 (q, 3J = 7.0 Hz, 2H), 3.70 (s, 3H), 3.50 (t, 3J = 6.8

Hz, 2H), 3.38 (dd, 2J = 14.0 Hz, 3J = 6.2 Hz, 1H), 3.22 (dd, 2J = 14.0 Hz, 3J = 6.2 Hz, 1H), 2.76 (t, 3J = 6.8 Hz, 2H), 1.26 (t, 3 J = 7.0 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 14.1, 19.7, 37.2, 38.4, 51.5, 53.8, 59.7, 129.9, 130.2, 136.3, 169.9, 171.7, 194.5 ppm. ESI-MS m/z [M + H]+, 356.10. [α]25 D = 14.5 ± 1.01 (c 0.1, MeOH). Ethyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-Lphenylalaninate (6.18). Yellow resin; yield, 82%; 1H-NMR (400 MHz, CDCl3): δH 7.51−7.05 (m, 5H), 5.45−5.38 (m, 1H), 4.20 (q, 3J = 7.1 Hz, 2H), 3.41 (dd, 2J = 14.0 Hz, 3J = 6.4 Hz, 1H), 3.16 (dd, 2J = 14.0 Hz, 3J = 6.4 Hz, 1H), 2.25 (s, 2H), 1.65 (s, 3H), 1.29 (s, 3H), 1.18 (s, 3H), 1.10 (t, 3J = 7.1 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 14.3, 20.4, 37.1, 38.6, 50.7, 53.9, 61.1, 130.0, 130.4, 136.5, 170.2, 172.7, 196.1 ppm. ESI-MS m/z [M + H]+, 368.13. [α]25 D = +11.7 ± 0.461 (c 0.1, MeOH). Isopropyl (((2-Cyanoethyl)thio)carbonothioyl)-L-phenylalaninate (6.19). Yellow resin; yield, 83%; 1H-NMR (400 MHz, CDCl3): δH 7.53−7.08 (m, 5H), 5.45−5.38 (m, 1H), 4.93− 4.84 (m, 1H), 4.14 (t, 3J = 7.1 Hz, 2H), 3.40 (dd, 2J = 14.0 Hz, 3 J = 6.6 Hz, 1H), 3.16 (dd, 2J = 14.0 Hz, 3J = 6.6 Hz, 1H), 2.93 (t, 3J = 7.1 Hz, 2H), 1.41 (d, 3J = 5.8 Hz, 6H) ppm. 13C-NMR (100 MHz, CDCl3): δC 20.1, 21.9, 37.1, 38.5, 54.7, 67.6, 119.8, 130.3, 130.6, 136.1, 170.8, 195.1 ppm. ESI-MS m/z [M + H]+, 336.11. [α]25 D = −7.80 ± 1.22 (c 0.1, MeOH). Isopropyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)1 L-phenylalaninate (6.20). Yellow resin; yield, 81%; H-NMR (400 MHz, CDCl3): δH 7.51−7.07 (m, 5H), 5.46−5.40 (m, 1H), 4.93−4.84 (m, 1H), 3.70 (s, 3H), 3.52 (t, 3J = 7.1 Hz, 2H), 3.38 (dd, 2J = 13.8 Hz, 3J = 6.4 Hz, 1H), 3.22 (dd, 2J = 13.8 Hz, 3J = 6.4 Hz, 1H), 2.76 (t, 3J = 7.1 Hz, 2H), 1.41 (d, 3J = 5.8 Hz, 6H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.8, 22.0, 37.2, 38.9, 51.5, 53.7, 70.1, 129.9, 130.2, 136.3, 169.9, 172.2, 195.1 ppm. ESI-MS m/z [M + H]+, 370.11. [α]25 D = −6.73 ± 0.902 (c 0.1, MeOH). Isopropyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-L-phenylalaninate (6.21). Yellow resin; yield, 79%; 1H-NMR (400 MHz, CDCl3): δH 7.51−7.05 (m, 5H), 5.45−5.38 (m, 1H), 4.93−4.84 (m, 1H), 3.32 (dd, 2J = 14.0 Hz, 3J = 6.4 Hz, 1H), 3.12 (dd, 2J = 14.0 Hz, 3J = 6.4 Hz, 1H), 2.18 (s, 2H), 1.69 (s, 3H), 1.51 (s, 3H), 1.42 (s, 3H), 1.30 (d, 3J = 5.8 Hz, 6H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.4, 21.9, 22.6, 30.2, 37.2, 52.9, 53.7, 68.2, 129.9, 130.2, 136.3, 170.2, 196.1, 209.2 ppm. ESI-MS m/z [M + H]+, 382.15. [α]25 D = −23.4 ± 0.529 (c 0.1, MeOH). Butyl (((2-Cyanoethyl)thio)carbonothioyl)-L-phenylalaninate (6.22). Yellow resin; yield, 91%; 1H-NMR (400 MHz, CDCl3): δH 7.54−7.04 (m, 5H), 5.45−5.38 (m, 1H), 4.16 (t, 3 J = 6.6 Hz, 2H), 3.41 (dd, 2J = 14.0 Hz, 3J = 6.5 Hz, 1H), 3.16 (dd, 2J = 14.0 Hz, 3J = 6.5 Hz, 1H), 2.93 (t, 3J = 6.8 Hz, 2H), 2.84 (t, 3J = 6.8 Hz, 2H), 1.67−1.55 (m, 2H), 1.41−1.29 (m, 2H), 0.94 (t, 3J = 7.4 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 13.7, 19.4, 19.7, 30.9, 37.3, 38.4, 54.2, 64.3, 119.2, 130.1, 130.2, 136.5, 170.4, 195.0. ESI-MS m/z [M + H]+, 350.12. [α]25 D = −2.60 ± 0.346 (c 0.1, MeOH). Butyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-Lphenylalaninate (6.23). Yellow resin; yield, 85%; 1H-NMR (400 MHz, CDCl3): δH 7.51−7.06 (m, 5H), 5.46−5.40 (m, 1H), 4.16 (t, 3J = 6.6 Hz, 2H), 3.71 (s, 3H), 3.52 (t, 3J = 6.8 Hz, 2H), 3.39 (dd, 2J = 14.1 Hz, 3J = 6.4 Hz, 1H), 3.22 (dd, 2J = 14.1 Hz, 3J = 6.4 Hz, 1H), 2.76 (t, 3J = 6.8 Hz, 2H), 1.62− 1.54 (m, 2H), 1.37−1.27 (m, 2H), 0.92 (t, 3J = 7.4 Hz, 3H)

3

H

DOI: 10.1021/acsomega.9b01098 ACS Omega XXXX, XXX, XXX−XXX

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ppm. 13C-NMR (100 MHz, CDCl3): δC 13.8, 19.3, 19.8, 30.9, 37.2, 38.6, 51.4, 53.8, 64.4, 129.9, 130.2, 136.3, 170.1, 172.0, 194.7 ppm. ESI-MS m/z [M + H]+, 384.12. [α]25 D = +5.47 ± 0.462 (c 0.1, MeOH). Butyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-Lphenylalaninate (6.24). Yellow resin; yield, 80%; 1H-NMR (400 MHz, CDCl3): δH 7.49−7.07 (m, 5H), 5.45−5.39 (m, 1H), 4.19 (t, 3J = 6.7 Hz, 2H), 3.32 (dd, 2J = 14.0 Hz, 3J = 6.2 Hz, 1H), 3.11 (dd, 2J = 14.0 Hz, 3J = 6.2 Hz, 1H), 2.18 (s, 2H), 1.65 (s, 3H), 1.55−1.48 (m, 2H), 1.30 (s, 3H), 1.24 (s, 3H), 1.19−1.13 (m, 2H), 0.94 (t, 3J = 7.4 Hz, 3H) ppm. 13CNMR (100 MHz, CDCl3): δC 13.9, 19.1, 19.9, 22.4, 30.2, 31.1, 37.3, 52.5, 52.9, 53.7, 64.2, 130.2, 130.1, 136.5, 170.1, 196.1, 209.0 ppm. ESI-MS m/z [M + H]+, 396.17. [α]25 D = −4.47 ± 0.306 (c 0.1, MeOH). Methyl (((2-Cyanoethyl)thio)carbonothioyl)-L-tyrosinate (6.25). Yellow resin; yield, 90%; 1H-NMR (400 MHz, CDCl3): δH 7.50 (bs, 1H), 6.92 (d, 3J = 8.4 Hz, 2H), 6.75 (d, 3J = 8.4 Hz, 2H), 5.42−5.36 (m, 1H), 3.76 (s, 3H), 3.49 (t, 3 J = 6.8 Hz, 2H), 3.31 (dd, 2J = 14.1 Hz, 3J = 5.9 Hz, 1H), 3.12 (dd, 2J = 14.1 Hz, 3J = 5.9 Hz, 1H), 2.82 (t, 3J = 6.8 Hz, 2H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.5, 38.2, 53.1, 54.3, 115.5, 119.1, 124.4, 130.4, 156.7, 170.2, 195.1 ppm. ESI-MS m/z [M + H]+, 324.08. [α]25 D = +16.9 ± 0.643 (c 0.1, MeOH) ppm. Methyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-Ltyrosinate (6.26). Yellow resin; yield, 89%; 1H-NMR (400 MHz, CDCl3): δH 7.44 (bs, 1H), 6.92 (d, 3J = 8.4 Hz, 2H), 6.74 (d, 3J = 8.4 Hz, 2H), 5.45−5.38 (m, 1H), 3.74 (s, 3H), 3.70 (s, 3H), 3.50 (t, 3J = 7.1 Hz, 2H), 3.32 (dd, 2J = 14.0 Hz, 3 J = 5.9 Hz, 1H), 3.10 (dd, 2J = 14.0 Hz, 3J = 5.9 Hz, 1H), 2.78 (t, 3J = 7.2 Hz, 2H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.8, 38.6, 51.4, 52.9, 53.8, 115.5, 124.4, 130.4, 156.7, 170.1, 172.0, 194.7 ppm. ESI-MS m/z [M + H]+, 358.09. [α]25 D = +25.1 ± 0.462 (c 0.1, MeOH) ppm. Methyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-L-tyrosinate (6.27). Yellow resin; yield, 80%; 1 H-NMR (400 MHz, CDCl3): δH 7.50 (bs, 1H), 6.93 (d, 3J = 8.0 Hz, 2H), 6.75 (d, 3J = 8.0 Hz, 2H), 5.45−5.38 (m, 1H), 3.75 (s, 3H), 3.32 (dd, 2J = 14.1 Hz, 3J = 5.9 Hz, 1H), 3.10 (dd, 2J = 14.1 Hz, 3J = 5.9 Hz, 1H), 2.17 (s, 2H), 1.63 (s, 3H), 1.25 (s, 3H), 1.11 (s, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.3, 22.4, 30.2, 52.6, 52.9, 53.5, 115.7, 124.6, 130.2, 156.8, 170.1, 196.2, 208.1 ppm. ESI-MS m/z [M + H]+, 370.11. [α]25 D = −23.9 ± 0.902 (c 0.1, MeOH) ppm. Ethyl (((2-Cyanoethyl)thio)carbonothioyl)-L-tyrosinate (6.28). Yellow resin; yield, 88%; 1H-NMR (400 MHz, CDCl3): δH 7.50 (bs, 1H), 6.92 (d, 3J = 8.5 Hz, 2H), 6.75 (d, 3J = 8.5 Hz, 2H), 5.35 (m, 1H), 4.20 (q, 3J = 7.1 Hz, 2H), 3.48 (t, 3J = 6.8 Hz, 2H), 3.34 (dd, 2J = 13.9 Hz, 3J = 5.9 Hz, 1H), 3.13 (dd, 2J = 13.9 Hz, 3J = 5.9 Hz, 1H), 2.81 (t, 3J = 6.8 Hz, 2H), 1.26 (t, 3J = 7.1 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 14.3, 19.8, 38.1, 54.4, 60.4, 115.5, 118.9, 124.4, 130.4, 156.7, 170.4, 194.8 ppm. ESI-MS m/z [M + H]+, 338.09. [α]25 D = +14.9 ± 0.902 (c 0.1, MeOH). Ethyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-L-tyrosinate (6.29). Yellow resin; yield, 91%; 1H-NMR (400 MHz, CDCl3): δH 7.43 (bs, 1H), 6.94 (d, 3J = 8.5 Hz, 2H), 6.73 (d, 3 J = 8.5 Hz, 2H), 5.41−5.35 (m, 1H), 4.19 (q, 3J = 7.0 Hz, 2H), 3.71 (s, 3H), 3.50 (t, 3J = 7.1 Hz, 2H), 3.32 (dd, 2J = 14.0 Hz, 3J = 5.9 Hz, 1H), 3.12 (dd, 2J = 14.0 Hz, 3J = 5.9 Hz, 1H), 2.76 (t, 3J = 7.1 Hz, 2H), 1.26 (t, 3J = 7.0 Hz, 3H) ppm. 13C-

NMR (100 MHz, CDCl3): δC 14.1, 19.7, 38.4, 51.5, 53.8, 59.7, 115.5, 124.4, 130.4, 156.7, 169.9, 171.7, 194.5 ppm. ESI-MS m/z [M + H]+, 372.10. [α]25 D = +16.9 ± 0.416 (c 0.1, MeOH). Ethyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-Ltyrosinate (6.30). Yellow resin; yield, 81%; 1H-NMR (400 MHz, CDCl3): δH 7.50 (bs, 1H), 6.93 (d, 3J = 8.5 Hz, 2H), 6.74 (d, 3J = 8.5 Hz, 2H), 5.43−5.39 (m, 1H), 4.19 (q, 3J = 7.1 Hz, 2H), 3.33 (dd, 2J = 13.9 Hz, 3J = 6.1 Hz, 1H), 3.12 (dd, 2J = 13.9 Hz, 3J = 6.1 Hz, 1H), 2.15 (s, 2H), 1.65 (s, 3H), 1.25 (s, 3H), 1.19 (s, 3H), 1.10 (t, 3J = 7.1 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 14.1, 20.1, 38.6, 50.8, 53.5, 60.7, 115.5, 124.6, 130.6, 156.7, 170.3, 172.3, 196.1 ppm. ESI-MS m/z [M + H]+, 384.13. [α]25 D = +4.53 ± 0.416 (c 0.1, MeOH). Isopropyl (((2-Cyanoethyl)thio)carbonothioyl)-L-tyrosinate (6.31). Yellow resin; yield, 89%; 1H-NMR (400 MHz, CDCl3): δH 7.50 (bs, 1H), 6.91 (d, 3J = 8.4 Hz, 2H), 6.78 (d, 3 J = 8.4 Hz, 2H), 5.42−5.36 (m, 1H), 4.86−4.78 (m, 1H), 3.49 (t, 3J = 7.1 Hz, 2H), 3.34 (dd, 2J = 14.0 Hz, 3J = 5.8 Hz, 1H), 3.12 (dd, 2J = 14.0 Hz, 3J = 5.8 Hz, 1H), 2.82 (t, 3J = 7.1 Hz, 2H), 1.52 (d, 3J = 5.8 Hz, 6H) ppm. 13C-NMR (100 MHz, CDCl3): δC 19.5, 21.7, 38.4, 54.5, 67.3, 115.5, 119.2, 124.4, 130.4, 156.7, 170.3, 195.2 ppm. ESI-MS m/z [M + H]+, 352.10. [α]25 D = −26.6 ± 0.346 (c 0.1, MeOH). Isopropyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)1 L-tyrosinate (6.32). Yellow resin; yield, 90%; H-NMR (400 3 MHz, CDCl3): δH 7.44 (bs, 1H), 6.92 (d, J = 8.4 Hz, 2H), 6.73 (d, 3J = 8.4 Hz, 2H), 5.45−5.38 (m, 1H), 4.86−4.76 (m, 1H), 3.74 (s, 3H), 3.50 (t, 3J = 7.2 Hz, 2H), 3.32 (dd, 2J = 14.0 Hz, 3J = 5.8 Hz, 1H), 3.11 (dd, 2J = 14.0 Hz, 3J = 5.8 Hz, 1H), 2.78 (t, 3J = 7.2 Hz, 2H), 1.52 (d, 3J = 5.8 Hz, 6H) ppm. 13CNMR (100 MHz, CDCl3): δC 19.5, 22.1, 38.9, 51.5, 53.9, 70.2, 115.7, 124.4, 130.6, 156.7, 169.9, 172.4, 195.4 ppm. ESI-MS m/z [M + H]+, 386.11. [α]25 D = +2.07 ± 0.115 (c 0.1, MeOH). Isopropyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-L-tyrosinate (6.33). Yellow resin; yield, 79%; 1 H-NMR (400 MHz, CDCl3): δH 7.49 (bs, 1H), 6.93 (d, 3J = 8.4 Hz, 2H), 6.73 (d, 3J = 8.4 Hz, 2H), 5.45−5.39 (m, 1H), 4.93−4.85 (m, 1H), 3.31 (dd, 2J = 14.0 Hz, 3J = 5.8 Hz, 1H), 3.12 (dd, 2J = 14.0 Hz, 3J = 5.8 Hz, 1H), 2.18 (s, 2H), 1.69 (s, 3H), 1.51 (s, 3H), 1.48 (s, 3H), 1.36 (d, 3J = 5.8 Hz, 6H) ppm. 13 C-NMR (100 MHz, CDCl3): δC 19.4, 21.9, 22.6, 30.2, 52.9, 53.7, 68.2, 115.5, 124.4, 130.4, 156.7, 170.2, 196.1, 209.1 ppm. ESI-MS m/z [M + H]+, 398.14. [α]25 D = −25.7 ± 0.416 (c 0.1, MeOH). Butyl (((2-Cyanoethyl)thio)carbonothioyl)-L-tyrosinate (6.34). Yellow resin; yield, 87%; 1H-NMR (400 MHz, CDCl3): δH 7.49 (bs, 1H), 6.92 (d, 3J = 8.2 Hz, 2H), 6.74 (d, 3J = 8.2 Hz, 2H), 5.42−5.35 (m, 1H), 4.18 (t, 3J = 6.6 Hz, 2H), 3.48 (t, 3J = 7.1 Hz, 2H), 3.34 (dd, 2J = 14.0 Hz, 3J = 6.1 Hz, 1H), 3.12 (dd, 2J = 14.0 Hz, 3J = 6.1 Hz, 1H), 2.87−2.75 (t, 3J = 7.1 Hz, 2H), 1.65−1.59 (m, 2H), 1.42−1.31 (m, 2H), 0.96 (t, 3J = 7.4 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 13.4, 19.5, 19.7, 31.2, 64.9, 38.7, 54.5, 115.7, 119.3, 124.4, 130.4, 156.7, 170.2, 195.1. ESI-MS m/z [M + H]+, 366.12. [α]25 D = −22.1 ± 1.15 (c 0.1, MeOH). Butyl (((3-Methoxy-3-oxopropyl)thio)carbonothioyl)-L-tyrosinate (6.35). Yellow resin; yield, 90%; 1H-NMR (400 MHz, CDCl3): δH 7.44 (bs, 1H), 6.93 (d, 3J = 8.4 Hz, 2H), 6.74 (d, 3 J = 8.4 Hz, 2H), 5.41−5.38 (m, 1H), 3.74 (s, 3H), 4.17 (t, 3J = 6.4 Hz, 2H), 3.49 (t, 3J = 7.2 Hz, 2H), 3.32 (dd, 2J = 14.0 Hz, 3J = 6.1 Hz, 1H), 3.11 (dd, 2J = 14.0 Hz, 3J = 6.1 Hz, 1H), 2.76 (t, 3J = 7.2 Hz, 2H), 1.69−1.58 (m, 2H), 1.43−1.29 (m, I

DOI: 10.1021/acsomega.9b01098 ACS Omega XXXX, XXX, XXX−XXX

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2H), 0.92 (t, 3J = 7.6 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 13.8, 19.3, 19.8, 30.9, 38.6, 51.4, 53.8, 64.4, 115.5, 124.4, 130.4, 156.7, 170.1, 172.0, 194.7 ppm. ESI-MS m/z [M + H]+, 400.12. [α]25 D = +7.07 ± 0.306 (c 0.1, MeOH). Butyl (((2-Methyl-4-oxopentan-2-yl)thio)carbonothioyl)-Ltyrosinate (6.36). Yellow resin; yield, 80%; 1H-NMR (400 MHz, CDCl3): δH 7.49 (bs, 1H), 6.92 (d, 3J = 8.4 Hz, 2H), 6.72 (d, 3J = 8.4 Hz, 2H), 5.45−5.38 (m, 1H), 4.18 (t, 3J = 6.6 Hz, 2H), 3.32 (dd, 2J = 14.0 Hz, 3J = 5.8 Hz, 1H), 3.10 (dd, 2J = 14.0 Hz, 3J = 5.8 Hz, 1H), 2.19 (s, 2H), 1.66 (s, 3H), 1.57− 1.48 (m, 2H), 1.31 (s, 3H), 1.26 (s, 3H), 1.18−1.12 (m, 2H), 0.92 (t, 3J = 7.4 Hz, 3H) ppm. 13C-NMR (100 MHz, CDCl3): δC 13.5, 19.2, 19.6, 22.6, 30.4, 31.1, 52.5, 52.8, 53.7, 64.4, 115.5, 124.6, 130.2, 156.4, 169.6, 195.4, 208.2 ppm. ESI-MS m/z: [M + H]+, 412.16. [α]25 D = +5.80 ± 0.721 (c 0.1, MeOH). Antifungal Assay. Compounds 5a−5l and 6.1−6.36 were assessed against F. oxysporum following the previously reported 12-well plate amended-medium method to evaluate the in vitro inhibition of mycelial growth.39 Mancozeb and iprodione were used as reference antifungals. Comparative Molecular Field Analysis (CoMFA). The 3D-QSAR analysis was performed using open3dqsar package adopting some parameters previously reported.49 Briefly, 3D structures of all compounds were built in Marvin (ChemAxon, Budapest, Hungary) and minimized by using SPARTAN ’14 (Wavefunction, Irvine, CA, USA) on a Dell Precision 7920 workstation. Geometries of compounds 5a−5l (precursors) and 6.1−6.36 (products) were fully optimized by using the AM1 method. Lowest-energy conformations were then considered as the bioactive conformations. AM1-derived molecular electrostatic potential distribution was used as net atomic charges. All the nitrogen and oxygen atoms were selected as tethers for substructure-based superimposing within the automatic alignment procedure. Thus, these tethers led us to superimpose all the compounds using an atom-by-atom least-squares fitting as an option implemented in Discovery Studio Client (Biovia, San Diego, CA, USA). The most active compound (i.e., 6.8) was selected as the reference molecule. Two subsets [i.e., test (30%) and training (70%) sets] were randomly generated from the set of aligned compounds. CoMFA descriptors, steric and electrostatic field energies, were then computed using default parameters: 2 A° grid point spacing, sp3 carbon probe atom (+1 charge), minimum column filtering of 2.0 kcal mol−1, and an energy cutoff of 30.0 kcal mol−1. The independent variable was the antifungal activity (expressed as pIC50 values). Leave-one-out (LOO) and leavemany-out (LMO) methods were employed for crossvalidations. The independent variable was predicted for the test set using high-quality validated models. Thus, calculations of the r2, cross-validated q2, and F-test values from the training dataset were used for the internally evaluation of the model robustness. Finally, a Y-scrambling procedure was implemented to demonstrate that the resulting model was not derived from a random correlation after 20 scramblings, 5 groups, and 10 runs, setting 0.85 as the critical point.50

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Lili Dahiana Becerra: 0000-0002-3366-022X Ericsson Coy-Barrera: 0000-0002-3553-9749 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present work is a product derived from the project INVCIAS-2043 funded by Vicerrectoriá de Investigaciones at UMNG−Validity 2016.

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Diego Quiroga: 0000-0001-5156-8200 J

DOI: 10.1021/acsomega.9b01098 ACS Omega XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsomega.9b01098 ACS Omega XXXX, XXX, XXX−XXX